The human mitochondrial proteome: oxidative stress, protein modifications and oxidative phosphorylation

Mitochondria are one of the most complex of subcellular organelles and play key roles in many cellular functions including energy production, fatty acid metabolism, pyrimidine biosynthesis, calcium homeostasis, and cell signaling. In recent years, we and other groups have attempted to identify the complete set of proteins that are localized to human mitochondria as a way to better understand its cellular functions and how it communicates with other cell compartment in complex signaling pathways such as oxidative stress and apoptosis. Indeed, there is an increasing interest in understanding the molecular details of oxidative stress and the mitochondrial role in this process, as well as assessing how mitochondrial proteins become damaged or posttranslationally modified as a consequence of a major change in a cell's redox status. In this review, we report on the current status of the human mitochondrial proteome with an emphasis towards understanding how mitochondrial proteins, especially the proteins that make up the respiratory chain or oxidative phosphorylation (OXPHOS) enzymes, are modified in various models of age-related diseases such as cancer and Parkinson's disease (PD).     

Control of mitochondria dynamics and oxidative metabolism by cAMP, AKAPs and the proteasome

Mitochondria are highly specialized organelles and major players in fundamental aspects of cell physiology. In yeast, energy metabolism and coupling of mitochondrial activity to growth and survival is controlled by the protein kinase A pathway. In higher eukaryotes, modulation of the so-called A-kinase anchor protein (AKAP) complex regulates mitochondrial dynamics and activity, adapting the oxidative machinery and the metabolic pathway to changes in physiological demand. Protein kinases and phosphatases are assembled by AKAPs within transduction units, providing a mechanism to control signaling events at mitochondria and other target organelles. Ubiquitin-mediated proteolysis of signal transducers and effectors provides an additional layer of complexity in the regulation of mitochondria homeostasis. Genetic evidence indicates that alteration of one or more components of these biochemical pathways leads to mitochondrial dysfunction and human diseases. In this review, we focus on the emerging role of AKAP scaffolds and the proteasome pathway in the control of oxidative metabolism, organelle dynamics and the mitochondrial signaling network. These aspects are crucial elements for maintaining a proper energy balance and cellular lifespan.     

c-Src kinase impairs the expression of mitochondrial OXPHOS complexes in liver cancer

Src family kinases (SFKs) play a crucial role in the regulation of multiple cellular pathways, including mitochondrial oxidative phosphorylation (OXPHOS). Aberrant activities of one of the most predominant SFKs, c-Src, was identified as a fundamental cause for dysfunctional cell signaling and implicated in cancer development and metastasis, especially in human hepatocellular carcinoma (HCC). Recent work in our laboratory revealed that c-Src is implicated in the regulation of mitochondrial energy metabolism in cancer. In this study, we investigated the effect of c-Src expression on mitochondrial energy metabolism by examining changes in the expression and activities of OXPHOS complexes in liver cancer biopsies and cell lines. An increased expression of c-Src was correlated with an impaired expression of nuclear- and mitochondrial-encoded subunits of OXPHOS complexes I and IV, respectively, in metastatic biopsies and cell lines. Additionally, we observed a similar association between high c-Src and reduced OXPHOS complex expression and activity in mouse embryonic fibroblast (MEF) cell lines. Interestingly, the inhibition of c-Src kinase activity with the SFK inhibitor PP2 and c-Src siRNA stimulated the expression of complex I and IV subunits and increased their enzymatic activities in both cancer and normal cells. Evidence provided in this study reveals that c-Src impairs the expression and function of mitochondrial OXPHOS complexes, resulting in a significant defect in mitochondrial energy metabolism, which can be a contributing factor to the development and progression of liver cancer. Furthermore, our findings strongly suggest that SFK inhibitors should be used in the treatment of HCC and other cancers with aberrant c-Src kinase activity to improve mitochondrial energy metabolism.     

Down-regulated energy metabolism genes associated with mitochondria oxidative phosphorylation and fatty acid metabolism in viral cardiomyopathy mouse heart

The majority of experimental and clinical studies indicates that the hypertrophied and failing myocardium are characterized by changes in energy and substrate metabolism that attributed to failing heart changes at the genomic level, in fact, heart failure is caused by various diseases, their energy metabolism and substrate are in different genetic variations, then the potential significance of the molecular mechanisms for the aetiology of heart failure is necessary to be evaluated. Persistent viral infection (especially coxsackievirus group B3) of the myocardium in viral myocarditis and viral dilated cardiomyopathy has never been neglected by experts. This study aimed to explore the role and regulatory mechanism of the altered gene expression for energy metabolism involved in mitochondrial oxidative phosphorylation, fatty acid metabolism in viral dilated cardiomyopathy. cDNA Microarray technology was used to evaluate the expression of >35,852 genes in a mice model of viral dilated cardiomyopathy. In total 1385 highly different genes expression, we analyzed 33 altered genes expression for energy metabolism involved in mitochondrial oxidative phosphorylation, fatty acid metabolism and further selected real-time-PCR for quantity one of regulatory mechanisms for energy including fatty acid metabolism—the UCP2 and assayed cytochrome C oxidase activity by Spectrophotometer to explore mitochondrial oxidative phosphorylation function. We found obviously different expression of 33 energy metabolism genes associated with mitochondria oxidative phosphorylation, fatty acid metabolism in cardiomyopathy mouse heart, the regulatory gene for energy metabolism: UCP2 was down-regulated and cytochrome C oxidase activity was decreased. Genes involved in both fatty acid metabolism and mitochondrial oxidative phosphorylation were down-regulated, mitochondrial uncoupling proteins (UCP2) expression did not increase but decrease which might be a kind of adaptive protection response to regulate energy metabolism for ATP produce.     

Epigallocatechin-3-gallate prevents oxidative phosphorylation deficit and promotes mitochondrial biogenesis in human cells from subjects with Down's syndrome

A critical role for mitochondrial dysfunction has been proposed in the pathogenesis of Down's syndrome (DS), a human multifactorial disorder caused by trisomy of chromosome 21, associated with mental retardation and early neurodegeneration. Previous studies from our group demonstrated in DS cells a decreased capacity of the mitochondrial ATP production system and overproduction of reactive oxygen species (ROS) in mitochondria. In this study we have tested the potential of epigallocatechin-3-gallate (EGCG) – a natural polyphenol component of green tea – to counteract the mitochondrial energy deficit found in DS cells. We found that EGCG, incubated with cultured lymphoblasts and fibroblasts from DS subjects, rescued mitochondrial complex I and ATP synthase catalytic activities, restored oxidative phosphorylation efficiency and counteracted oxidative stress. These effects were associated with EGCG-induced promotion of PKA activity, related to increased cellular levels of cAMP and PKA-dependent phosphorylation of the NDUFS4 subunit of complex I. In addition, EGCG strongly promoted mitochondrial biogenesis in DS cells, as associated with increase in Sirt1-dependent PGC-1α deacetylation, NRF-1 and T-FAM protein levels and mitochondrial DNA content.In conclusion, this study shows that EGCG is a promoting effector of oxidative phosphorylation and mitochondrial biogenesis in DS cells, acting through modulation of the cAMP/PKA- and sirtuin-dependent pathways. EGCG treatment promises thus to be a therapeutic approach to counteract mitochondrial energy deficit and oxidative stress in DS.     

Mechanisms of mitochondrial response to variations in energy demand in eukaryotic cells

This review focuses on the different mechanisms involved in the adjustment of mitochondrial ATP production to cellular energy demand. The oxidative phosphorylation steady state at constant mitochondrial enzyme content can vary in response to energy demand. However, such an adaptation is tightly linked to a modification in both oxidative phosphorylation yield and phosphate potential and is obviously very limited in eukaryotic cells. We describe the three main mechanisms involved in mitochondrial response to energy demand. In heart cells, a short-term adjustment can be reached mainly through metabolic signaling via phosphotransfer networks by the compartmentalized energy transfer and signal transmission. In such a complex regulatory mechanism, Ca²⁺ signaling participates in activation of matricial dehydrogenases as well as mitochondrial ATP synthase. These processes allow a large increase in ATP production rate without an important modification in thermodynamic forces. For a long-term adaptation, two main mechanisms are involved: modulation of the mitochondrial enzyme content as a function of energy demand and/or kinetic regulation by covalent modifications (phosphorylations) of some respiratory chain complex subunits. Regardless of the mechanism involved (kinetic regulation by covalent modification or adjustment of mitochondrial enzyme content), the cAMP signaling pathway plays a major role in molecular signaling, leading to the mitochondrial response. We discuss the energetic advantages of these mechanisms. yeast; C6 glioma cells; muscle; kinetic regulation     

Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation

The quantity and activity of mitochondria vary dramatically in tissues and are modulated in response to changing cellular energy demands and environmental factors. The amount of mitochondrial DNA (mtDNA), which encodes essential subunits of the oxidative phosphorylation complexes required for cellular ATP production, is also tightly regulated, but by largely unknown mechanisms. Using murine T cells as a model system, we have addressed how specific signaling pathways influence mitochondrial biogenesis and mtDNA copy number. T cell receptor (TCR) activation results in a large increase in mitochondrial mass and membrane potential and a corresponding amplification of mtDNA, consistent with a vital role for mitochondrial function for growth and proliferation of these cells. Independent activation of protein kinase C (via PMA) or calcium-related pathways (via ionomycin) had differential and sub-maximal effects on these mitochondrial parameters, as did activation of naïve T cells with proliferative cytokines. Thus, the robust mitochondrial biogenesis response observed upon TCR activation requires synergy of multiple downstream signaling pathways. One such pathway involves AMP-activated protein kinase (AMPK), which we show has an unprecedented role in negatively regulating mitochondrial biogenesis that is mammalian target of rapamycin (mTOR)-dependent. That is, inhibition of AMPK after TCR signaling commences results in excessive, but uncoordinated mitochondrial proliferation. Thus mitochondrial biogenesis is not under control of a single master regulatory circuit, but rather requires the convergence of multiple signaling pathways with distinct downstream consequences on the organelle’s structure, composition, and function.     

Impaired mitochondrial respiration promotes dendritic branching via the AMPK signaling pathway

Functional neuronal circuits require a constant remodeling of their network composed of highly interconnected neurons. The plasticity of synapses and the shaping of elaborated dendritic branches are energy demanding and therefore depend on an efficient mitochondrial oxidative phosphorylation (OXPHOS). The spatial and functional regulations of dendritic patterning occur also after cell fate specification; however, the molecular mechanisms underlying this complex process remain elusive. Here, we exploit the changes in dendritic architecture in highly branched neurons as a result of aberrant mitochondrial activity. In sensory neurons of Caenorhabditis elegans, genetic manipulations of mitochondrial complex I subunits cause an unexpected outgrowth of dendritic arbors and ectopic structures. The increased number of dendritic branches is coordinated through a specific signaling cascade rather than as a simple consequence of oxidative stress. On the basis of genetic and pharmacological evidence, we show that OXPHOS deficiency promotes branching through the activation of the AMP-activated protein kinase AMPK and the downstream target phosphoinositide 3-kinase PI3K. Taken together, our findings describe a well-defined signaling pathway that regulates dendritic outgrowth in conditions of compromised OXPHOS and the resulting AMPK activation.     

Implication of liver cardiolipins in mitochondrial energy metabolism disorder in cancer cachexia

Mitochondrial membranes are essential for the good functioning of the organelle. For instance, the inner mitochondrial membrane contains the oxidative phosphorylation system that permits ATP synthesis. Phospholipids environment and especially cardiolipin are crucial for the mitochondrial energy metabolism. Indeed, cardiolipin is known to provide essential structural and functional support to several proteins involved in oxidative phosphorylation. Alterations in cardiolipin structure, content and fatty acids composition have been associated with mitochondrial dysfunction in several physiopathological conditions and diseases. Cancer cachexia is a complex and dynamic process characterized by a negative energy balance induced by anorexia and hypermetabolism which leads to a drastic loss in body weight that aggravate prognosis of cancer patients. The underlying mechanisms of hypermetabolism are not fully understood. Whether the mitochondrial energy metabolism is altered during this disease and may participate to hypermetabolism is not clear. This mini-review focuses on cardiolipin especially its biosynthesis and remodeling pathways, its relation with mitochondrial energy metabolism and its possible implication in the cancer cachexia syndrome.     

Loss of major nutrient sensing and signaling pathways suppresses starvation lethality in electron transport chain mutants

The electron transport chain (ETC) is a well-studied and highly conserved metabolic pathway that produces ATP through generation of a proton gradient across the inner mitochondrial membrane coupled to oxidative phosphorylation. ETC mutations are associated with a wide array of human disease conditions and to aging-related phenotypes in a number of different organisms. In this study, we sought to better understand the role of the ETC in aging using a yeast model. A panel of ETC mutant strains that fail to survive starvation was used to isolate suppressor mutants that survive. These suppressors tend to fall into major nutrient sensing and signaling pathways, suggesting that the ETC is involved in proper starvation signaling to these pathways in yeast. These suppressors also partially restore ETC-associated gene expression and pH homeostasis defects, though it remains unclear whether these phenotypes directly cause the suppression or are simply effects. This work further highlights the complex cellular network connections between metabolic pathways and signaling events in the cell and their potential roles in aging and age-related diseases.     

The H-ATP synthase: A gate to ROS-mediated cell death or cell survival

Cellular oxidative stress results from the increased generation of reactive oxygen species and/or the dysfunction of the antioxidant systems. Most intracellular reactive oxygen species derive from superoxide radical although the majority of the biological effects of reactive oxygen species are mediated by hydrogen peroxide. In this contribution we overview the major cellular sites of reactive oxygen species production, with special emphasis in the mitochondrial pathways. Reactive oxygen species regulate signaling pathways involved in promoting survival and cell death, proliferation, metabolic regulation, the activation of the antioxidant response, the control of iron metabolism and Ca^2 + signaling. The reversible oxidation of cysteines in transducers of reactive oxygen species is the primary mechanism of regulation of the activity of these proteins. Next, we present the mitochondrial H⁺-ATP synthase as a core hub in energy and cell death regulation, defining both the rate of energy metabolism and the reactive oxygen species-mediated cell death in response to chemotherapy. Two main mechanisms that affect the expression and activity of the H⁺-ATP synthase down-regulate oxidative phosphorylation in prevalent human carcinomas. In this context, we emphasize the prominent role played by the ATPase Inhibitory Factor 1 in human carcinogenesis as an inhibitor of the H⁺-ATP synthase activity and a mediator of cell survival. The ATPase Inhibitory Factor 1 promotes metabolic rewiring to an enhanced aerobic glycolysis and the subsequent production of mitochondrial reactive oxygen species. The generated reactive oxygen species are able to reprogram the nucleus to support tumor development by arresting cell death. Overall, we discuss the cross-talk between reactive oxygen species signaling and mitochondrial function that is crucial in determining the cellular fate. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca²⁺, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD⁺] ratio, which was corroborated by using the NADH/NAD⁺ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD⁺ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD⁺ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.     

Protein phosphorylation and prevention of cytochrome oxidase inhibition by ATP: coupled mechanisms of energy metabolism regulation

Rapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intramitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation. Using protein mutagenesis, molecular dynamics simulations, and induced fit docking, we show that mitochondrial energy metabolism regulation by phosphorylation of COXIV-1 is coupled with prevention of COX allosteric inhibition by ATP. This regulatory mechanism is essential for efficient oxidative metabolism and cell survival. We propose that S58 COXIV-1 phosphorylation has evolved as a metabolic switch that allows mammalian mitochondria to rapidly toggle between energy utilization and energy storage.     

c-Jun N-terminal kinase regulates mitochondrial bioenergetics by modulating pyruvate dehydrogenase activity in primary cortical neurons

This study examines the role of c- jun N-terminal kinase (JNK) in mitochondrial signaling and bioenergetics in primary cortical neurons and isolated rat brain mitochondria. Exposure of neurons to either anisomycin (an activator of JNK/p38 mitogen-activated protein kinases) or H₂O₂ resulted in activation (phosphorylation) of JNK (mostly p46^JNK1) and its translocation to mitochondria. Experiments with mitochondria isolated from either rat brain or primary cortical neurons and incubated with proteinase K revealed that phosphorylated JNK was associated with the outer mitochondrial membrane; this association resulted in the phosphorylation of the E_1α subunit of pyruvate dehydrogenase, a key enzyme that catalyzes the oxidative decarboxylation of pyruvate and that links two major metabolic pathways: glycolysis and the tricarboxylic acid cycle. JNK-mediated phosphorylation of pyruvate dehydrogenase was not observed in experiments carried out with mitoplasts, thus suggesting the requirement of intact, functional mitochondria for this effect. JNK-mediated phosphorylation of pyruvate dehydrogenase was associated with a decline in its activity and, consequently, a shift to anaerobic pyruvate metabolism: the latter was confirmed by increased accumulation of lactic acid and decreased overall energy production (ATP levels). Pyruvate dehydrogenase appears to be a specific phosphorylation target for JNK, for other kinases, such as protein kinase A and protein kinase C did not elicit pyruvate dehydrogenase phosphorylation and did not decrease the activity of the complex. These results suggest that JNK mediates a signaling pathway that regulates metabolic functions in mitochondria as part of a network that coordinates cytosolic and mitochondrial processes relevant for cell function.     

Inhibition of mitochondrial protein synthesis influences the glucocorticoid sensitivity of lymphoid cells

Inhibition of mitochondrial protein synthesis impairs the formation of the 13 polypeptides encoded on the mitochondrial genome. These polypeptides are part of enzyme complexes involved in oxidative phosphorylation. Prolonged inhibition of mitochondrial protein synthesis thus reduces the oxidative phosphorylation capacity which ultimately results in impairment of energy-requiring processes. Via a different mechanism glucocorticoid hormones also decrease the oxidative phosphorylation capacity of, e.g., lymphoid cells. The present study shows that inhibition of mitochondrial protein synthesis influences glucocorticoid-induced responses of lymphoid cells in two opposing manners. (a) It is enhanced after induction in cells with a reduced oxidative phosphorylation capacity resulting from preceding inhibition of mitochondrial protein synthesis. This can be explained by the synergistic effects of glucocorticoids and prolonged inhibition of mitochondrial protein synthesis on energy-producing processes. (b) It is counteracted when mitochondrial protein synthesis is impaired during induction of the response. The latter observation suggests that mitochondrial protein synthesis is involved in the generation of glucocorticoid-induced effects on lymphoid cells.